Proteases are responsible either directly or indirectly for all bodily functions including cell growth, nutrition, differentiation and apoptosis. They also play a significant role in intracellular and extracellular protein turn over (house keeping and repair), cell migration and invasion, fertilization and implantation (Protease inhibitors, novel therapeutic application and development, Tony E Hugli, TIBTECH, 14, 409-412, 1996). Since proteases are necessary for normal and abnormal body functions, their effective regulatory counterparts i.e., protease inhibitors, are tremendously essential for physiological regulations. Protease inhibitors have been the source of attention in many disciplines. Due to their presence in valuable plant feeds and involvement in nutritive properties they have evoked the interest of nutritionists. Inhibitor proteins have been studied for the elucidation of mechanism of inhibition of proteases, as well as for the studies on protein-protein interactions and associations. Due to their unique pharmacological properties, protease inhibitors are also used as valuable tools in medical research.
Protease inhibitors are classified into Synthetic and Naturally occurring inhibitors. They occur in numerous animal tissues and fluids, in many plant tissues (particularly in legume seeds and other storage organs) and in microorganisms (Protease inhibitors, Yehudith Birk, Hydrolytic enzymes, A Neuberger and K. Brocklehurst (Eds), Elsevier Science Publishers B. V. (Biomedical Division), 257, 1987). The most abundant source of the inhibitors in plants is the seeds, but their location is not necessarily restricted to this part of the plant. They are also found in leaves, tubers, etc. As for the intracellular localization of the inhibitors, they appear to be associated primarily with the cytosol, but in some instances they have been localized in protein bodies. The inhibitors of animal origin are found both in tissues and in secretions of organs. The pancreatic trypsin inhibitor has been found as an intracellular component in various bovine organs: in the pancreas, lung, liver, spleen, paratoid gland and also in pituitary gland. In addition to the thoroughly studied pancreatic trypsin inhibitors, a large number of protease inhibitors from different animal sources have been isolated. Many of them are secretory proteins, such as trypsin inhibitors of blood plasma, milk colustrum, seminal plasma and submandibular glands. The plasma protease inhibitors constitute a major group of the functional proteins of the blood plasma. Most of them inhibit serine proteases but their mechanism of interaction is still being actively pursued by many investigators.
The presence of protease inhibitors in microorganisms came into existence from the studies on antibiotics as they act as inhibitors of the enzymes which are involved in growth and multiplication. Proteolytic enzymes outside of microbial cells hydrolyze organic nitrogen compounds in the medium, so they are thought to be harmful to cells. The production of inhibitors of the proteolytic enzymes by microorganisms is probably a mechanism to provide cell protection. In contrast to the inhibitors of proteolytic enzymes obtained from animals and plants, the inhibitors from microorganisms are of smaller molecular nature. Specific inhibitors of microbial origin have been used as useful tools in biochemical analysis of biological functions and diseases. (Enzyme inhibitors of microbial origin, Hamao Umezawa, University Park Press).
A few of the inhibitors of microbial origin of therapeutic interest are given below:
Leupeptin-from Streptomyces, is the inhibitor of trypsin, plasmin, kalikrein and papain.
Chymostatin-from Streptomyces, is the inhibitor of chymotrypsin.
Dopastin-from Pseudomonas, Oosponol-from Oospora, Oudenone-from Oudemansialla radicata and Fusaric acid-from Fusarium are the inhibitors of dopamine hydroxylase.
Pepstatin A-from Streptomyces, is the inhibitor of pepsin, an aspartic acid protease. It inhibits the HIV-1 protease, which is also an aspartic protease and the key enzyme for the propagation of the HIV.
The expanding Acquired Immuno Deficiency Syndrome (AIDS) epidemic and the relentless nature of the disease have intensified the search for effective antiviral therapies, to control the replication of the HIV, the causative agent of AIDS. The HIV-1 protease is the key enzyme for the propagation of the virus. Thus specific inhibition of the HIV-1 protease by inhibitors is useful in preventing the infection HIV-1 protease is structurally and mechanistically related to mammalian and microbial aspartic proteases such as pepsin, cathepsin, renin, and endothiopepsin. The classification of HIV-1 protease in the aspartyl family was also predicted from its primary sequence analysis. A highly conserved sequence Asp-Thr-Gly (D-T-G) in retroviral proteases, is also conserved in the active site of the cellular and fungal proteases. Molecular modeling studies have also confirmed the functional and structural similarities of the retroviral proteases to other aspartyl proteases. Various synthetic peptide and non-peptide compounds have been shown to inhibit HIV-1 protease. Well documented examples of isolation of compounds by microbial screening represented by the discovery of potent compounds such as cyclosprin, movionolin and avermycin, etc. An antifungal antibiotic cerulenin from Cephalosporium and pepstatin A, a pepsin inhibitor from Streptomyces, have been well characterized as HIV-1 protease inhibitors (C.Debouck, AIDS Research and Human Retroviruses, 8, 153-164, 1992).
Extensive evidence suggests that, the degradation of hemoglobin is necessary for the growth of erythrocytic malarial parasite, apparently to provide free amino acids for parasitic protein synthesis. On the basis of the data available, the aspartic acid proteases are thought to be responsible for the initial cleavages of hemoglobin. Both aspartic acid and cystein proteases have synergistic effects in inhibiting the growth of the cultural malarial parasite and also these proteases act synergistically to degrade hemoglobin. Therefore, the combination of inhibitors of malarial cystein and aspartic acid proteases, may provide a most effective chemotherapeutic regimen and best limit the development of parasitic resistant to protease inhibitors. Pepstatin, the inhibitor of aspartic acid proteases, along with the cystein protease inhibitor E-64, blocks the Plasmodium falciparum development. (Proteases of Malarial Parasite: New Targets for Chemotherapy, Philip J. Rosenthal, Emerging Infectious Diseases, 4(1), 49-57, 1998). So far no report is available for the preparation of the protease inhibitor using alkalothermophilic Bacillus sp.
Based on the fact that the aspartic acid protease plays a significant role in the development of the malarial parasite, the applicants believe that the inhibitor produced in accordance with the practice of the invention using the novel strain of Bacillus sp deposited at ATCC having Accession No. PTA 972, could be a potent inhibitor for proteases, particularly aspartic acid protease, and more particularly for proteases of malarial parasites. The inhibitor described in the present invention inhibits pepsin, an aspartic protease. Pepsin present in the gastric secretion is responsible for the degradation (digestion) of proteinaceous food. Excess secretion of pepsin has harmful effects on the stomach as it damages the digestive tract and causes stomach ulcer or duodenal ulcer. Considering the fact that the inhibitor is an active inhibitor of pepsin, it has potential application as a therapeutic agent against stomach or duodenal ulcers. Pepstatin A, a pepsin inhibitor has been reported to inhibit HIV-1 protease which is also an aspartic protease. The applicants have observed that the inhibitor also inhibits other enzymes having aspartic acid in the active site, and felt that the microbial protease inhibitor could inhibit HIV-1 protease.